The long term goal remains a detailed understanding of how the Na/K pump works and how it may be modulated. The Na/K pump plays the vital role of maintaining the electrochemical gradients for Na and K ions that underlie electrical signaling, essential coupled transport, and cell pH and volume regulation; the Na/K pump is also the receptor for the still widely prescribed cardiotonic steroid, and pump inhibitor, digoxin. Charge translocation is a fundamental feature of the ion pumping cycle, and of individual partial reactions. It provides a readily accessible, reproducible, and sensitive signal for assaying turnover rates and rates of conformational transitions, and sheds light on the molecular mechanism of ion transport, now viewed in light of the new high-resolution crystal structure of the related SR Ca pump. Specific aim (1) is to further investigate the ion transport mechanism, using two approaches. In one, we will continue characterizing the charge translocating steps by quantitative analysis of the dependence on membrane potential, external and internal ion and nucleotide concentrations, and temperature, of steady- and pre-steady-state pump currents in internally dialyzed guinea-pig ventricular myocytes and squid giant axons (in which technical advances now permit ultra high-speed measurements of pump-mediated charge movements, resolving relaxation rates as fast as 10 to the 5th power per s, some 3 orders of magnitude faster than the Na/K pump's maximum turnover rate. In the other, the lethal coral toxin, palytoxin, is used to transform the Na/K pump into a gated ion channel. We will express in HEK293 cells mutant ouabain-resistant Na/K pumps with cysteine residues introduced at strategic locations, and then use sulfhydrl-specific reagents to investigate structure of the gates and mechanisms of gating, which should provide information on ion occlusion/deocclusion mechanisms during normal Na/K pumping. Specific aim (2) is to see whether, under what conditions, and by which mechanisms, Na/K pump activity in myocytes may be acutely modulated by cellular regulatory processes like kinase-mediated phosphorylation of the pump (or associated regulatory molecule), or interactions with cytoplasmic Ca ions. We will directly apply regulatory molecules, such as purified kinases or phosphatases, to the pump's cytoplasmic surface in giant inside-out patches of membrane, excised from myocytes. Explicit kinetic models of the Na/K transport mechanism will be developed to account for experimental observations, and will be refined by fits to the data.